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Team:University of Lethbridge/Project
From 2008.igem.org
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The “Bacuum Cleaner” – an intelligent self-propelling keener cleaner
Our goal is to create a modified Escherichia coli bacterium capable of seeking out and degrading toxic aromatic pollutants created during the oil refinery and mining processes. Our “bacuum cleaner” will respond to this destructive compound through interaction with a programmed riboswitch (an mRNA capable of binding a target molecule with its 5’ UTR which causes a conformational change, thereby regulating any downstream genes). By using promoters of differential strength, we can alter the signal induced by varying concentrations of target ligand. At low concentrations, we intend to have our riboswitch activate the motility protein cheZ in E. coli, thus directing the bacterium towards higher concentrations of our target molecule (i.e. positive chemotaxis). Once it reaches a threshold concentration, a catabolic pathway capable of degrading our target pollutant will be activated.
We plan to use SELEX to reprogram the theophylline riboswitch initially characterized by last year’s team, aiming to have it bind one of the many toxic aromatics produced. This not only builds on our past work with riboswitches but also demonstrates an alternative function for riboswitch-controlled chemotaxis, changing from a simple detection to active “search and destroy” role. Fluorescent protein expression will used to demonstrate and characterize the functionality of the riboswitch _______ details details details etc.
Our project also has major environmental implications, especially in Alberta, where the oil industry is the driving economic sector. Not only will our “bacuum” remove these compounds from an aqueous system, they also will eliminate them, going a step further than the ordinary less-intelligent bag-dependent vacuum cleaner. Ultimately we aim to have our bacteria convert the target aromatic into a useable compound. One example would be the biosynthesis of fatty acids as an alternative fuel source, creating a “bioreactor” as a possible aid to another serious environmental dilemma.
Project Details
Part 1: Chemotaxis - the "search" from "search and destroy"
Our subproject is to ensure our 'bacuum cleaner' will search out and move towards our potential target. Using a riboswitch which responds to this target, a gene involved in motility in Escherichia coli (CheZ) will be switched to an 'on' state. CheZ controls bacterial movement by dephosphorylating CheY, altering the bacteria from random tumbling to directed movement. By doing so, we hope to observe the bacteria moving towards the target molecule in a positive chemotaxis manner.
Because we wish to eventually have our bacteria degrade this activating compound, we need to have a multi-level response system established. Our bacteria should respond to low levels of the target by activating our CheZ riboswitch (through a strong binding aptamer) and to high levels by activating the catabolic pathway responsible for its degradation. By doing so, we will have created our 'search and destroy' system.
Our subgroup will attempt to establish a working motility assay to prove control of chemotaxis is possible with an already characterized theophylline-binding riboswitch and later with our new target molecule. We will also use a colour read-out system of two fluorescent proteins to demonstrate the differential binding of the ligand at varying concentrations.
Part 2
Part 3
Part 4
Results
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